¹³C and ²H Analysis of Fatty Acid Methyl Esters (FAMEs)

Isotope-Ratio Mass Spectrometry
Fatty acids are made suitable for gas chromatography isotope-ratio mass spectrometry following derivatization as fatty acid methyl esters (FAMEs) [1,2]. Column selection for CSIA of FAMEs is determined by sample composition: a nonpolar DB-5ms UI capillary column (60 m x 0.25 mm ID x 1 mm film thickness) is used for soils or oils analysis, while a highly polar DB-FATWAX UI capillary column (30 m x 0.25 mm ID x 0.25 mm film thickness) for animal tissues and most marine samples.

For soil and oil samples, FAMEs are dissolved in heptane, injected at 290 ºC (splitless, 1 min), and separated on an Agilent DB-5ms UI column (60 m x 0.25 mm ID x 1 µm film thickness) at constant flow rate of 1.2 mL/min under the following temperature program: 80 °C (hold 1 min); 220 °C (4 °C/min); 290 °C (10 °C/min; hold 30 min.).

For animal and marine samples, FAMEs are dissolved in heptane, injected at 220 ºC (splitless, 1 min), and separated on an Agilent DB-FATWAX UI capillary column (30 m x 0.25 mm ID x 0.25 mm film thickness) at constant flow rate of 1.2 mL/min under the following temperature program: 60 °C (hold 1 min); 100 °C (5 °C/min); 175 °C (2 °C/min; hold 10 min.); 220 °C (2 °C/min; hold 20 min.).

GC-C-P-IRMS is performed on a Thermo Trace GC 1310 gas chromatograph coupled to a Thermo Finnigan MAT 253 isotope-ratio mass spectrometer via a GC IsoLink II combustion interface. For ¹³C analysis, individual FAMEs are converted to CO₂ within a combustion reactor composed of a NiO tube containing CuO/NiO wires maintained at 1000 °C. Water is subsequently removed through a nafion dryer and the analyte gases transferred to the IRMS. For ²H analysis, individual FAMEs are converted to H₂ within a high-temperature thermal conversion reactor of graphitized Al₂O₃ tube maintained at 1425 °C.

One of every five samples are analyzed in duplicate. Replicates of the quality control and assessment materials are measured every 5 samples.

Fig.1. Chromatogram of δ¹³C analysis of fatty acid methyl esters from fish muscle lipids.

UCD SIF FAMEs Compound List*

* VLCFAs, up to c32:0, and other FAMEs not listed here may be measured with prior consultation.

Calibration and Reporting of Stable Isotope Ratios

Quality control and assessment mixtures are composed of pure FAMEs (or FAs) that have been calibrated separately by EA- and TC/EA-IRMS using certified reference materials (e.g. NBS-22, IAEA-CH-7) distributed by NIST (Gaithersburg, MD U.S.A.) or the USGS (Reston, VA U.S.A.) and are directly traceable to the primary isotopic reference material for each element (δ¹³C: V-PDB; δ²H: V-SMOW). All calibration procedures for CSIA of FAMEs are applied identically across reference and sample materials. First, the provisional isotopic value for each FAME is obtained by normalization to an isotopically-calibrated internal reference compound (e.g. c12:0, c13:0). Isotopic values of the individual FAMEs are then scale-normalized to the primary reference material (δ¹³C: V-PDB; δ²H: V-SMOW) using an external mixture composed of FAMEs with a broad range of calibrated δ¹³C and δ²H values. Through each calibration step, FAMEs mixtures and secondary QA materials are monitored for accuracy and precision. Final quality assessment and acceptance of analysis is based on the accuracy and precision of the unbiased quality assessment materials.

Acceptance or rejection of calibrated data is based on the accuracy and precision of an unbiased quality assurance material, a second δ¹³C- and δ²H-calibrated FAMEs mixture. Acceptance of sample measurements require that the final calibrated isotopic values and mean standard deviation (σ) of the quality assurance replicates fall within expected measurement error ( less than ±0.5 ‰). Precision estimates from the co-measured calibrated FAMEs mixtures and quality assurance materials are provided with data reports.

Measurement Uncertainty

Sample materials are inherently variable in fatty acid composition. Some FAMEs may not be measureable and measurement error among FAMEs varies between different sample types due to differences in composition and chromatographic resolution. Accuracy and precision of the co-measured calibrated FAMEs mixtures and quality assurance materials are provided with data reports. Limit of quantification, based on total peak area, is generally 1 V-s for δ¹³C and > 10 V-s for δ²H.

References

[1] K. Eder. 1995. Gas chromatographic analysis of fatty acid methyl esters. Journal of Chromatography B 671: 113-131. doi: 10.1016/0378-4347(95)00142-6

[2] W. Meier-Augenstein. 2002. Stable isotope analysis of fatty acids by gas chromatography-isotope ratio mass spectrometry. Analytica Chimica Acta 465: 63-79. doi: 10.1016/S0003-2670(02)00194-0